The lignocellulosic biomass forms a large reserve, as yet underused, of fermentable sugars. These sugars may be obtained by hydrolysis of cellulose and of hemicelluloses present in a large amount in said biomass.
Processes for hydrolysing cellulose and hemicelluloses have already been proposed. An older one is chemical hydrolysis, generally performed by means of strong acids. The industrial development of such a process is difficult. More recently, numerous studies have been conducted which have led to enzymatic hydrolysis processes offering the advantage of avoiding degradation of potential sugars present in the biomass. Such a technology, in order to be used on industrial scale, requires large amounts of cellulases. A systematic experimentation has led to the discovery of a continuous process for manufacturing cellulases under efficient and cheap conditions, whereby the enzymatic hydrolysis of the lignocellulosic biomass may be considered as industrially feasible.
Amongst various microorganism known to excrete cellulases, the Trichoderma reesei ascomycete fungus is considered as one of the more adapted to an industrial production of theses enzymes: Mandels, Annual Reports on fermentation processes vol. 5, G. Tsao Editor, Academic Press N.Y. 1981, p. 35-78.
The wild strains of this fungus have the capacity to excrete, in the presence of cellulose, the mixture of all the enzymatic activities subsequently required for hydrolysing the lignocellulosic biomass. The resultant cellulases have exo- and endo-glucanase, .beta.-glucosidease, exo- and endo-xylanase and .beta.-xylosidase activity. The main prior works concern improvements to wild strains by conventional genetic techniques of mutation-selection, which led to the formation of hyperproducing strains of Trichoderma reesei, such as MCG 77 (deposited at the Northern Regional Research Center, Ill., under serial number NRRL 11, 236) disclosed by Gallo in U.S. Pat. No. 4,275,163; MCG 80 disclosed by Allen, A. L. and Andreotti, R. E., Biotechnology and bioengineering Symposium No.12, pages 451-459 (1982); RUT C 30 disclosed by Montenecourt, B. S. and Eveleigh, D. E., Applied Environmental Microbiology, volume 34, page 777 (1977) and CL 847 disclosed By Warzywoda, M., at al, Biotechnology Letter, volume 5, page 243 (April 1983).
This genetic improvement has provided strains having a maximum productivity of excreted cellulases as well as a decreased catabolic repression as compared to that of the wild strain. This catabolic repression is a determinant factor in the production of cellulases on industrial scale; as a matter of fact, the synthesis of cellulases is blocked by the presence, in the culture medium, of easily metabolizable sugars. The various above-mentioned strains have been the object of research and development work which have confirmed their superiority as compared to the wild strain for cellulase excretion. Mandels, above cited, has shown that, as for the wild strain, higher enzyme productions are obtained by feeding these strains with purified celluloses as the main source of energy and carbon. Under these conditions, the cellulase production is proportional to the amount of cellulose added to the medium, at least as far as this cellulose does not result in too high a viscosity of the medium, which no longer allows normal efficiency in the different transfers required for the cultivation. It is thus accepted that the maximum achievable concentration of cellulose is about 70-80 g/l for example. These culture media of high purified cellulose content cannot be extrapolated to industrial scale in view, on the one hand, of the high cost of purified cellulose and, on the other hand, of the high power consumption for stirring culture media of very high viscosity.
Searches for carbon sources providing easier extrapolation for the culture media have thus been made. Allen, A. L. and Mortensen, R. E., Biotechnology and Bioengineering, volume 23, pages 2641-2645 (1981) have proposed as carbon sources several sugars used alone or as mixtures, in an view of their low cost and their high solubility in aqueous medium. However, it is observed that the cellulase production obtained by cultivating Trichoderma reesei on culture media based on soluble sugars is much lower than that obtained on cellulose, particularly on purified cellulose. This low production is probably attributable to a repressing effect of sugars, at the prevailing concentrations, on the production of enzymes.
Techniques for the continuous feeding of a carbonaceous substrate have been used to feed microorganisms with the sugar amounts strictly required for their needs, and to limit the residual sugar content in the cultures, thus preventing the catabolic repression due to sugars. The use of these techniques with non soluble cellulosic substrates raises the problem of extrapolation under sterile conditions. Thus attempts have been made to use these techniques with soluble sugars whose concentrated solutions may be easily sterilized and introduced under sterile conditions by pumping or by any other liquid transfer means into reactors of variable capacity. Allen and Andreotti, above mentioned, have used a source of lactose, soluble sugar, as carbon source for the strain MCG 80 Trichoderma reesei, whereas Allen and Mortensen, above-mentioned, have used glucose, cellobiose or mixtures of artificial sugars with the strain RUT C 30 Trichoderma reesei. The results obtained by these authors, concerning the content, the yield as well as the productivity of cellulase in continuous operation, although higher than those obtained in batch on the same substrates, do not reach however those obtained in batch on purified celluloses.